The World Health Organization declared that new antibiotics are urgently needed to treat carbapenem resistant pathogens. Moreover, mortality from multi-drug resistant (MDR) bacterial infections is projected to cause 10 million deaths per year worldwide by 2050, making antibiotic resistance a serious threat. Despite the need for novel drugs, pharmaceutical companies have dropped research and development interests, primarily due to low profitability; thus, antibiotic discovery is of utmost importance. Pseudomonas aeruginosa is a versatile opportunistic pathogen notorious for its role in cystic fibrosis (CF) patients in which a chronic lung infection leads to a life threating disease, and future effective treatment of MDR P. aeruginosa infections will require novel, yet undiscovered, antibiotics. While much research has been devoted to the pathogenicity of P. aeruginosa, understanding its natural lifecycle should provide insights into important aspects contributing to its susceptibility, as many CF-derived P. aeruginosa originate from ecological isolates. P. aeruginosa strains are repeatedly observed to represent an extreme minority of environmentally-derived Pseudomonas populations (i.e., closely related isolates that cluster as distinct phylogenetic groups), suggesting a decreased fitness of P. aeruginosa in habitats that are distinct from a CF lung. Instead, environments are dominated by other pseudomonads, whose global abundance and omnipresence suggest the expression of certain traits that are advantageous to ecological survival. A trait that is likely to contribute to such fitness effects is the ability to antagonize nearby competitors through production of antimicrobial factors. Here, the research team proposes that in environments where multiple groups of closely related pseudomonads are in sustained competition, such antagonistic interactions lead to decreased fitness of other isolates, such as P. aeruginosa, that are better adapted to and more fit in a human host. Thus, investigating direct interactions between environmental bacteria and MDR pathogens should provide the means to identify effective, and novel, antimicrobial factors. Indeed, recent data from the PI shows that pseudomonads inhibit MDR P. aeruginosa including isolates with carbapenem resistant. Using an innovative and rigorous culture- based methodology, the hypothesis will be tested by (i) identifying pseudomonads that inhibit CF-derived MDR pathogens, (ii) characterizing the biosynthetic gene clusters involved in antimicrobial activity, and (iii) genome analysis of antagonistic strains and biochemical characterization of novel compounds. Results will contribute to detailed characterization of natural antibiotics produced by environmental bacteria that are effective against MDR pathogens.